LOCUS       sequence-451485-        5555 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MCMUbHis.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5555)
  AUTHORS   Williams C, Dong KC, Arkinson C, Martin A
  TITLE     The Ufd1 cofactor determines the linkage specificity of 
            polyubiquitin chain engagement by the AAA+ ATPase Cdc48.
  JOURNAL   Mol Cell. 2023 Mar 2;83(5):759-769.e7. doi: 
            10.1016/j.molcel.2023.01.016. Epub 2023 Feb 2.
  PUBMED    36736315
REFERENCE   2  (bases 1 to 5555)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.molcel.2023.01.016"; journalName: "Mol Cell"; date: 
            "2023-03-2- 2"; volume: "83"; issue: "5"; pages: "759-769.e7"
COMMENT     Sequence Label: pAM299
FEATURES             Location/Qualifiers
     source          1..5555
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(8..29)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     220..239
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(363..410)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     406..424
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(477..494)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(550..567)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(568..591)
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             complement(592..819)
                     /codon_start=1
                     /product="Saccharomyces cerevisiae ubiquitin"
                     /label=ubiquitin
                     /translation="MQIFVKTLTGKTITLEVESSDTIDNVKSKIQDKEGIPPDQQRLIF
                     AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG"
     RBS             835..857
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    870..894
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     complement(894..913)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(895..913)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(997..1016)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1222..1299
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1300..2382
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(1319..1338)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3191..3382
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(3376..3398)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3484..3623
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3539..3558
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3638..3655)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3809..4397)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3889..3908)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(4508..4527)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             4519..5334
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             4519..5334
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(4592..4611)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(join(5426..5555,1..326))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
ORIGIN
        1 tgttgttcca gtttggaaca agagtccact attaaagaac gtggactcca acgtcaaagg
       61 gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa ccatcaccct aatcaagttt
      121 tttggggtcg aggtgccgta aagcactaaa tcggaaccct aaagggagcc cccgatttag
      181 agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa gggaagaaag cgaaaggagc
      241 gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc gtaaccacca cacccgccgc
      301 gcttaatgcg ccgctacagg gcgcgtccca ttcgccaatc cggatatagt tcctcctttc
      361 agcaaaaaac ccctcaagac ccgtttagag gccccaaggg gttatgctag ttattgctca
      421 gcggtggcag cagccaactc agcttccttt cgggctttgt tagcagccgg atctcagtgg
      481 tggtggtggt ggtgctcgag tgcggccgca agcttgtcga cggagctcga attcggatcc
      541 tcatcattag tggtgatgat ggtgatgggg cccctggaac agaacttcca gaccacctct
      601 tagccttagc acaagatgta aggtggactc cttctgaatg ttgtaatcag acagcgttct
      661 accgtcttct agctgcttac cggcaaagat caatctttgt tgatctggag ggataccttc
      721 cttgtcttga attttcgact taacgttgtc gatggtatcg gaagattcaa cttccaatgt
      781 tatggtttta ccggtcaaag tcttgacgaa aatctgcata cacatatggg tatatctcct
      841 tcttaaagtt aaacaaaatt attcagaggg gaattgttat ccgctcacaa ttcccctata
      901 gtgagtcgta ttaatttcgc gggatcgaga tctcgatcct ctacgccgga cgcatcgtgg
      961 ccggcatcac cggcgccaca ggtgcggttg ctggcgccta tatcgccgac atcaccgatg
     1021 gggaagatcg ggctcgccac ttcgggctca tgagcgcttg tttcggcgtg ggtatggtgg
     1081 caggccccgt ggccggggga ctgttgggcg ccatctcctt gcatgcacca ttccttgcgg
     1141 cggcggtgct caacggcctc aacctactac tgggctgctt cctaatgcag gagtcgcata
     1201 agggagagcg tcgagatccc ggacaccatc gaatggcgca aaacctttcg cggtatggca
     1261 tgatagcgcc cggaagagag tcaattcagg gtggtgaatg tgaaaccagt aacgttatac
     1321 gatgtcgcag agtatgccgg tgtctcttat cagaccgttt cccgcgtggt gaaccaggcc
     1381 agccacgttt ctgcgaaaac gcgggaaaaa gtggaagcgg cgatggcgga gctgaattac
     1441 attcccaacc gcgtggcaca acaactggcg ggcaaacagt cgttgctgat tggcgttgcc
     1501 acctccagtc tggccctgca cgcgccgtcg caaattgtcg cggcgattaa atctcgcgcc
     1561 gatcaactgg gtgccagcgt ggtggtgtcg atggtagaac gaagcggcgt cgaagcctgt
     1621 aaagcggcgg tgcacaatct tctcgcgcaa cgcgtcagtg ggctgatcat taactatccg
     1681 ctggatgacc aggatgccat tgctgtggaa gctgcctgca ctaatgttcc ggcgttattt
     1741 cttgatgtct ctgaccagac acccatcaac agtattattt tctcccatga agacggtacg
     1801 cgactgggcg tggagcatct ggtcgcattg ggtcaccagc aaatcgcgct gttagcgggc
     1861 ccattaagtt ctgtctcggc gcgtctgcgt ctggctggct ggcataaata tctcactcgc
     1921 aatcaaattc agccgatagc ggaacgggaa ggcgactgga gtgccatgtc cggttttcaa
     1981 caaaccatgc aaatgctgaa tgagggcatc gttcccactg cgatgctggt tgccaacgat
     2041 cagatggcgc tgggcgcaat gcgcgccatt accgagtccg ggctgcgcgt tggtgcggat
     2101 atctcggtag tgggatacga cgataccgaa gacagctcat gttatatccc gccgttaacc
     2161 accatcaaac aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc
     2221 tctcagggcc aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa
     2281 accaccctgg cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg
     2341 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt
     2401 aagttagctc actcattagg caccgggatc tcgaccgatg cccttgagag ccttcaaccc
     2461 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     2521 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     2581 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     2641 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     2701 cattatcgcc ggcatggcgg ccccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     2761 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     2821 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     2881 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     2941 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     3001 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     3061 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     3121 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     3181 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     3241 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     3301 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg
     3361 tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta
     3421 agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg
     3481 gggcgcagcc atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg
     3541 gcatcagagc agattgtact gagagtgcac cattgcggtg tgaaataccg cacagatgcg
     3601 taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3661 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3721 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3781 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3841 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3901 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3961 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4021 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4081 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4141 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4201 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
     4261 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4321 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4381 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4441 acgaaaactc acgttaaggg attttggtca tgaacaataa aactgtctgc ttacataaac
     4501 agtaatacaa ggggtgttat gagccatatt caacgggaaa cgtcttgctc taggccgcga
     4561 ttaaattcca acatggatgc tgatttatat gggtataaat gggctcgcga taatgtcggg
     4621 caatcaggtg cgacaatcta tcgattgtat gggaagcccg atgcgccaga gttgtttctg
     4681 aaacatggca aaggtagcgt tgccaatgat gttacagatg agatggtcag actaaactgg
     4741 ctgacggaat ttatgcctct tccgaccatc aagcatttta tccgtactcc tgatgatgca
     4801 tggttactca ccactgcgat ccccgggaaa acagcattcc aggtattaga agaatatcct
     4861 gattcaggtg aaaatattgt tgatgcgctg gcagtgttcc tgcgccggtt gcattcgatt
     4921 cctgtttgta attgtccttt taacagcgat cgcgtatttc gtctcgctca ggcgcaatca
     4981 cgaatgaata acggtttggt tgatgcgagt gattttgatg acgagcgtaa tggctggcct
     5041 gttgaacaag tctggaaaga aatgcataaa cttttgccat tctcaccgga ttcagtcgtc
     5101 actcatggtg atttctcact tgataacctt atttttgacg aggggaaatt aataggttgt
     5161 attgatgttg gacgagtcgg aatcgcagac cgataccagg atcttgccat cctatggaac
     5221 tgcctcggtg agttttctcc ttcattacag aaacggcttt ttcaaaaata tggtattgat
     5281 aatcctgata tgaataaatt gcagtttcat ttgatgctcg atgagttttt ctaagaatta
     5341 attcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
     5401 cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg
     5461 ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct
     5521 tataaatcaa aagaatagac cgagataggg ttgag
//
